One impediment to developing an effective vaccine for HIV infection is the requirement for a vaccination method that leads to strong immune responses. DNA vaccines have potential, but they are often poor immunogens unless an adjuvant plasmid is added. Experiments are described that show that plasmid DNA for a new soluble, multimeric form of CD40L is an effective adjuvant for DNA vaccines in mouse models, and it will be important to test other immunostimulatory members of the TNF superfamily (TNFSF). Combining Toll-like Receptor (TLR) and CD40 stimulation is highly synergistic, and will be tested using the novel form of CD40L above. Additionally, microencapsulation of DNA vaccines with PLGA/PBAE copolymer helps to carry the DNA to dendritic cells and also upregulates CD40, indicating that it might synergize with CD40L as an adjuvant. Consequently, this project has the following aims: Aim 1: To test the individual effects of plasmids for OX40L, RANKL, CD27L, and LIGHT on HIV DNA vaccination in mice. Aim 2: To test combinations of CD40L and bacterial lipopeptide (BLP) TLR2 agonists as DNA vaccine adjuvants. Aim 3: To determine if DNA microencapsulation with PLGA/PBAE synergizes with CD40L to enhance the immune response to an HIV DNA vaccine. Aim 4: To prepare macaque versions of the best TNFSF constructs for advancement to the SIV/macaque model. The innovative aspects of this R21 proposal are: its use of a promising new delivery platform for CD40L and other TNFSFs; studies of CD40L plus BLPs as DNA vaccine adjuvants; and combining CD40L with PLGA/PBAE microencapsulation in an HIV DNA vaccine. In sum, these studies will optimize a powerful adjuvant platform for HIV DNA vaccines that has great potential for DNA-only vaccination or DNA prime/viral vector boost protocols. In addition, this DNA vaccine approach may prove generally applicable for emerging infections and other diseases for which a potent vaccine is needed.